Isolated porcine pancreatic cells for use in treatment of diseases characterized by insufficient insulin activity

ABSTRACT

Isolated porcine pancreatic cells, isolated populations of such cells and methods for isolating and using the cells to treat subjects with diseases characterized by insufficient insulin activity are described. The porcine pancreatic cells are preferably non-insulin-secreting porcine pancreatic cell having the ability to differentiate into an insulin-secreting cell upon introduction into a xenogeneic subject, such as a human subject. Such cells include embryonic porcine pancreatic cells obtained from embryonic pigs between about day 31 and day 35 of gestation. The porcine pancreatic cells can be modified to be suitable for transplantation into a xenogeneic subject, for example, by altering an antigen (e.g., an MHC class I antigen) on the cell surface which is capable of stimulating an immune response against the cell in the subject (e.g., by contact with an anti-MHC class I antibody, or a fragment or derivative thereof). The isolated porcine pancreatic cells of the invention can be used to treat diseases characterized by insufficient insulin activity, e.g., Type I and Type II diabetes, by administering the cells to a subject having such a disease.

BACKGROUND OF THE INVENTION

Idiopathic or primary diabetes mellitus is a chronic disorder ofcarbohydrate, fat, and protein metabolism characterized in its fullyexpressed form by an absolute or relative insulin deficiency, fastinghyperglycemia, glycosuria, and a striking tendency toward development ofatherosclerosis, microangiopathy, nephropathy, and neuropathy.Underutilization of glucose is characteristic of all diabetic patients,but only some have a clearly defined severe insulin deficiency resultingfrom a loss of β cells. The large remainder of diabetic patients sufferfrom some impairment of insulin secretory response associated with amarked resistance to insulin in the peripheral tissues.

The phrase "idiopathic diabetes mellitus" embraces a heterogeneous groupof disorders having in common the above-described characteristics. Atleast two major as well as several less common variants of the diseasehave been identified. One major variant, insulin-dependent diabetesmellitus (IDDM) (Type I), accounts for about 10% of diabetics. A secondmajor variant, non-insulin-dependent diabetes mellitus (NIDDM) (Type II)represents the remaining 90% of all diabetic patients. Robbins, S. L. etal. Pathologic Basis of Disease, 3rd Edition (W.B. Saunders Company,Philadelphia, 1984) p. 972. Absent regular insulin replacement therapyusing exogenously produced insulin and/or careful monitoring of the dietof diabetic patients, such patients experience a wide range ofdebilitating symptoms, some of which can progress into coma andultimately death.

An alternative method of treating diabetes presently underinvestigation, which does not require repeated administration of insulinand/or strict monitoring of diet, is transplantation of pancreatic cellsor tissue from a donor to the diabetic patient. A major problem inpancreatic cell or tissue transplantation from one human to another fortreatment of diabetes, however, is a shortage of donor tissue. Thompson,S. C. et al. (1990) Transplantation 49(3): 571-581. Moreover, humanpancreas will inevitably remain in limited supply and be subject to manyconstraints, including, especially with human fetal pancreatic cells ortissue, sensitive ethical issues. The improvement in patients and graftsurvival following human pancreas transplants (Sutherland, D. E. R. etal. (1987) Transplant. Proc. 19: 113) will also mean that adultcadaveric pancreas may be more difficult to obtain for experimentalpurposes.

As a result of the above-described problems associated withtransplantation of pancreatic tissue from a human donor to a humanrecipient, alternative sources of pancreatic tissue for transplantationhave been investigated. Several groups of investigators have conductedresearch involving the use of pancreatic cells and tissue from animalsources, such as swine, for transplantation. See e.g., Korsgren, O. etal. (1993) Surgery 113: 205-214; Braesch, M. K. et al. (1992)Transplant. Proc. 24(2): 679-680; Groth, C. G. et al. (1992) Transplant.Proc. 24(3): 972-973; Liu, X. et al. (1991) Diabetes 40: 858-866;Korsgren, O. et al. (1991) Diabetologia 34: 379-386; Yoneda, K. et al.(1989) Diabetes 38 (Supp. 1): 213-216; Wilson, J. D. et al. (1989)Diabetes 38 (Suppl. 1): 217-219; Korsgren, O. et al. (1989) Diabetes 38(Suppl. 1): 209-212; Korsgren, O. et al. (1988) Transplantation 45(3):509-514; Sasaki, N. et al. (1984) Transplantation 38(4): 335-340.Several of these investigators report transplantation of pancreatictissue samples containing insulin-secreting and at least partiallydifferentiated porcine pancreatic cells into xenogeneic subjects aftershort-term culture. However, these short-term cultures of pancreaticcells often display eventual necrosis (Thompson, S. C. et al. (1990)Transplantation 49(3): 571-581), developmental stagnation (Liu, X. etal. (1991) Diabetes 40: 858-866; Korsgren, O. et al. (1989) Diabetes 38(Suppl. 1): 209-212; Korsgren, O. et al. (1988) Transplantation 45(3):509-514), decreased proliferation (Liu, X. et al. (1991) Diabetes 40:858-866; Korsgren, O. et al. (1988) Transplantation 45(3): 509-514), anddecreased insulin production (Yoneda, K. et al. (1989) Diabetes 38(Supp. 1): 213-216; Korsgren, O. et al. (1988) Transplantation 45(3):509-514).

SUMMARY OF THE INVENTION

The present invention provides porcine pancreatic cell(s) which can beused to generate populations of cells useful for transplantation intodiabetic subjects. The porcine pancreatic cells of the invention arecapable of proliferating in vitro and in vivo and are insulin-secretingafter transplantation into a recipient subject. Accordingly, theinvention pertains to isolated non-insulin-secreting porcine pancreaticcells having the ability to differentiate into insulin-secreting cellsupon introduction into a xenogeneic subject. In one embodiment, thenon-insulin-secreting porcine pancreatic cells are embryonic pancreaticcells isolated during certain stages of gestational development. It hasbeen discovered that such porcine embryonic pancreatic cells can bemaintained in culture if sub-confluent and will proliferate for longperiods of time, e.g., six months or more, without forming pseudoislet-like aggregates. Preferably, the pancreatic cells are obtainedfrom embryonic pigs at an early stage of development (i.e., prior toformation of islets in vivo) and are maintained in culture to allow cellproliferation without substantial differentiation into islet-likeaggregates. Moreover, the culture is not diluted out bynon-insulin-producing cells, e.g., endothelial cells, which are involvedin islet formation. Furthermore, proliferation of the culturedpancreatic cells can be substantially augmented by adding certainembryonic proliferating agents to the culture. These embryonicproliferating agents can decrease the doubling time of the cells byalmost two-fold. When the cells are allowed to reach confluence, theybegin to form pseudo islet-like aggregates which produce insulin,glucagon, and somatostatin.

Thus, large populations of non-insulin-secreting porcine pancreaticcells capable of proliferating and differentiating to produceinsulin-secreting cells upon introduction into a subject can be preparedin an economical and time-efficient manner. The porcine pancreatic cellsof the invention can, therefore, serve as a convenient and plentifulsource of cells for administration to subjects having diseases caused byinsufficient activity of a pancreatic hormone, e.g, insulin, e.g., TypeI or Type II diabetes, or enzyme.

Accordingly, the instant invention pertains to an isolated porcinepancreatic cell and a population of porcine pancreatic cells suitablefor administration to a xenogeneic subject, particularly a humansubject. The isolated porcine pancreatic cell, alone or in a population,produces glucagon and somatostatin in certain embodiments, but does notsecrete insulin. Upon introduction into a xenogeneic subject, however,the porcine pancreatic cell proliferates and differentiates to form apopulation of insulin-secreting cells. Preferred porcine pancreaticcells are embryonic porcine pancreatic cells obtained from an embryonicpig at a selected gestational age. The preferred gestational age ofembryonic swine from which to obtain pancreatic cells suitable fortransplantation into xenogeneic subjects, particularly humans, wasdetermined to be between about twenty (28) and about forty (40) days,more preferably between about thirty (30) and thirty-five (35) days,most preferably between about thirty-one (31) and about thirty-five (35)days of gestation. It is preferred that the porcine pancreatic cells beobtained from a pig which is essentially free from organisms orsubstances which are capable of transmitting infection or disease to axenogeneic recipient of the cells as described herein. Typically, theporcine pancreatic cells are isolated from a pig which is essentiallyfree from at least one organism selected from the group consisting ofparasites, bacteria, mycoplasma, and viruses. In addition, the porcinepancreatic cells can be modified as described herein. The porcinepancreatic cells of the invention can be grown as a cell culture in amedium suitable to support the growth of the cells. In addition, theporcine pancreatic cells can be inserted into a delivery device, e.g., asyringe, which facilitates the introduction of the cells into a subject.

The invention further pertains to a porcine pancreatic cell and anisolated population of such cells which, in unmodified form, have atleast one antigen on the cell surface which is capable of stimulating animmune response against the cell(s) in a xenogeneic subject, forexample, a human. The antigen on the surface of the porcine pancreaticcell(s) is altered to inhibit rejection of the cell(s) when introducedinto a xenogeneic subject. In one embodiment, the cell surface antigenwhich is altered is an MHC class I antigen. This MHC class I antigen canbe contacted, prior to transplantation into a xenogeneic subject with atleast one MHC class I antibody, or a fragment or derivative thereof,which binds to the MHC class I antigen on the cell surface but does notactivate complement or induce lysis of the cell. One example of an MHCclass I antibody is an MHC class I F(ab')₂ fragment, such as an MHCclass I F(ab')₂ fragment of a monoclonal antibody PT85. In oneembodiment, the porcine pancreatic cells are obtained from embryonicpigs of the preferred gestational ages described herein. Porcinepancreatic cells to be modified in this manner can be obtained from apig which is essentially free from organisms or substances which arecapable of transmitting infection or disease to a xenogeneic recipientof the cells as described herein.

A further aspect of the invention pertains to methods of promoting orinducing proliferation of embryonic porcine pancreatic cells in whichthe cells are contacted with at least one embryonic proliferating agentwhich promotes or induces proliferation of the cells in vitro and/or invivo. Preferred embryonic proliferating agents for promoting or inducingproliferation of embryonic porcine pancreatic cells includeplatelet-derived growth factor (PDGF) and epidermal growth factor (EGF).Other embryonic proliferating agents include growth factors for whichthe embryonic porcine pancreatic cells of a certain gestational age(e.g., between about 31 and 35 days of gestation) express a receptor.

The invention also provides methods of isolating and promotingproliferation of porcine pancreatic cells in vitro prior to theadministration of the cells to a xenogeneic subject. These methodstypically include isolating porcine pancreatic cells from an embryonicpig and contacting the cells with an embryonic proliferating agent, suchas PDGF, EGF or growth factors for which the embryonic porcinepancreatic cells express a receptor, which promotes proliferation of thecells. The cells are preferably isolated from an embryonic pig fromabout day 31 to about day 35 of gestation. In one embodiment the porcinepancreatic cells are non-insulin-secreting cells which have the abilityto differentiate into insulin-secreting cells upon introduction into axenogeneic subject. The cells can be administered to the xenogeneicsubject prior to or after in vitro formation of insulin-secretingislet-like aggregates.

The invention still further provides methods of treating diseasescharacterized by insufficient insulin activity, e.g, Type I and Type IIdiabetes, in a subject, e.g., a human, having such a disease. In oneembodiment, a subject having the disease is administered an amount of apopulation of non-insulin-secreting porcine pancreatic cells which areobtained from an embryonic pig, e.g., cells from an embryonic pigbetween about day 31 and day 35 of gestation, having the ability todifferentiate into insulin-secreting cells following administration tothe subject. These cells can be modified as described herein prior tointroduction into the subject. In another embodiment, a subject having adisease characterized by insufficient insulin activity is administered apopulation of modified porcine pancreatic cells of the invention or apopulation of porcine pancreatic cells obtained from pigs which areessentially free from organisms and substances which are capable oftransmitting infection or disease to the subject. These methods canfurther include the step of administering an immunosuppressive agent tothe subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depict insulin staining of fetal pig pancreatic cells inculture. FIG. 1A is a phase micrograph of fetal pig pancreatic cells inmonolayer prior to formation of islet-like clusters. No insulin stainingcan be detected in these cells at this time. FIG. 1B is a phasemicrograph of fetal pig pancreatic cells that were allowed to formislet-like clusters (indicated by arrows) and stained positive forinsulin.

FIGS. 2A-2B depict grafts obtained from fetal pig pancreatic cellstransplanted into nude mice. FIG. 2A is a section from transplantedkidney showing the graft stained with aldehyde-fuchsin.Insulin-containing cells stain darkly and can be seen scatteredthroughout the graft. FIG. 2B is a section from the same graft stainedwith antibodies which recognize insulin. Again, the insulin-containingcells within the graft stain more darkly than the other cells. In bothfigures, the insulin-positive cells are indicated by arrows, the donorgraft is marked (G), and the recipient mouse kidney is marked (K).

DETAILED DESCRIPTION OF THE INVENTION I. Isolated Cells and CellPopulations of the Invention

A. Non-Insulin-Secreting Porcine Pancreatic Cells Suitable forAdministration to Xenogeneic Subjects

This invention features an isolated non-insulin-secreting porcinepancreatic cell having the ability to differentiate into aninsulin-secreting cell upon introduction into a xenogeneic subject.These cells can be used to treat diseases, such as Type I and Type IIdiabetes, which are characterized by insufficient activity of thehormones, e.g., insulin, and enzymes produced by the pancreas. As usedherein, the term "isolated" refers to a cell which has been separatedfrom its natural environment. This term includes gross physicalseparation from its natural environment, e.g., removal from the donoranimal, e.g., a pig, and alteration of the cell's relationship with theneighboring cells with which it is in direct contact by, for example,dissociation. Isolation does not refer to a cell which is in a tissuesection, is cultured as part of a tissue section, or is transplanted inthe form of a tissue section. When used to refer to a population ofporcine pancreatic cells, the term "isolated" includes populations ofcells which result from proliferation of the isolated cells of theinvention.

When isolated from a donor swine, the pancreatic cells of the inventionare non-insulin-secreting. The phrase "non-insulin-secreting" refers tocells which do not deposit detectable or therapeutically significantamounts of insulin into their surroundings, e.g., tissue fluid orculture medium. Therapeutically significant amounts of insulin includeamounts which are capable of reducing or alleviating at least oneadverse effect or symptom of a disease characterized by insufficientinsulin activity when an appropriate number of the insulin-secretingcells are introduced in a subject having such a disease. However, theisolated cells have the ability to differentiate into insulin-secretingcells in vitro or upon introduction into a subject. In one embodiment,the non-insulin-secreting porcine pancreatic cells are furthercharacterized by the ability to produce detectable amounts of glucagonand/or somatostatin upon isolation from the donor swine. The pancreaticcells of the invention, when administered to a xenogeneic subject,"proliferate", a term which is used herein to mean reproduce ormultiply, to produce or form a population i.e., a group of two or morecells. Differentiation, as used herein, refers to cells which haveacquired functions different from and/or in addition to those that thecells originally possessed. For example, an non-insulin porcinepancreatic cell can differentiate, under specific conditions, into acell which secretes insulin. As used herein, the phrase "secreteinsulin" refers to cells which deposit insulin into their surroundings,e.g., tissue fluid, e.g, blood, or culture medium. A common method foranalyzing tissue fluid or culture media for insulin-secretion is byradioimmunoassay. See Heding, L. G. (1972) Diabetologia 8: 260.

The term "subject" is intended to include mammals, particularly humans,susceptible to diseases characterized by insufficient insulin activity.The term "subject" also includes mammals in which an immune response iselicited against allogeneic or xenogeneic cells. Examples of subjectsinclude primates (e.g., humans, and monkeys). A "xenogeneic subject"(also referred to herein as "recipient subject" or "recipient") as usedherein is a subject into which cells of another species are introducedor are to be introduced.

The pancreas is a mixed exocrine and endocrine gland. The exocrineportion is composed of several serous cells surrounding a lumen. Thesecells synthesize and secrete digestive enzymes such as trypsinogen,chymotrypsinogen, carboxypeptidase, ribonuclease, deoxyribonuclease,triacylglycerol lipase, phospholipase A₂, elastase, and amylase. Theendocrine portion of the pancreas is composed of the islets ofLangerhans. The islets of Langerhans appear as rounded clusters of cellsembedded within the exocrine pancreas. Four different types of cells- α,β, δ, and φ-have been identified in the islets. The α cells constituteabout 20% of the cells found in pancreatic islets and produce thehormone glucagon. Glucagon acts on several tissues to make energyavailable in the intervals between feeding. In the liver, glucagoncauses breakdown of glycogen and promotes gluconeogenesis from aminoacid precursors. The δ cells produce somatostatin which acts in thepancreas to inhibit glucagon release and to fiecrease pancreaticexocrine secretion. The hormone pancreatic polypeptide is produced inthe φ cells. This hormone inhibits pancreatic exocrine secretion ofbicarbonate and enzymes, causes relaxation of the gallbladder, anddecreases bile secretion. The most abundant cell in the islets,constituting 60-80% of the cells, is the β cell, which produces insulin.Insulin is known to cause the storage of excess nutrients arising duringand shortly after feeding. The major target organs for insulin are theliver, muscle, and fat-organs specialized for storage of energy.

The language "pancreatic cell" refers to a cell which can produce ahormone or enzyme normally produced by a pancreatic cell, e.g., an atleast partially differentiated α, β, δ, or φ cell, and a cell, e.g., apancreatic precursor cell, which can develop into a cell which canproduce a hormone or enzyme normally produced by a pancreatic cell. Inone embodiment, the porcine pancreatic cells are characterized by theability to produce glucagon and/or somatostatin upon isolation from adonor swine. The pancreatic cells of the invention can also be culturedprior to administration to a subject under conditions which promote cellproliferation and differentiation. These conditions include culturingthe cells to allow proliferation and confluence in vitro at which timethe cells form pseudo islet-like aggregates or clusters and secreteinsulin, glucagon, and somatostatin.

Pancreatic cells of the invention are obtained from the pancreas of adonor swine (also referred to herein as "pig") such as, for example, aswine which is essentially pathogen-free as described herein. In apreferred embodiment, the pancreatic cells are obtained from theprimordial pancreas (also referred to herein as "fetal pancreas" and"embryonic pancreas") of an embryonic donor swine and preferably at aselected gestational age. The selected gestational ages (the totalgestation time for pig is approximately 115 days) for obtainingprimordial pancreatic cells are determined based on the followingcriteria: the ability of the embryonic porcine pancreas structure to beidentified; the viability of the cells upon isolation from the donorpig, the ability of the cells to proliferate in culture; the ability ofthe cells to remain undifferentiated (i.e., non-insulin secreting) inculture; and the ability of the cells to differentiate (i.e., secretepancreatic hormones, e.g., insulin, and enzymes) upon introduction intoa recipient subject. The preferred gestational age of embryonic swinefrom which to obtain pancreatic cells suitable for introduction intoxenogeneic subjects, particularly humans, was determined to be betweenabout twenty (28) and about forty (40) days, more preferably aboutthirty (30) and about thirty-five (35) days, most preferably aboutthirty-one (31) and about thirty-five (35) days of gestation. Earlierthan about days 28-30 of gestation, the primordial pancreas in embryonicswine is not as easy to identify. Later than about days 35-36 ofgestation, the pancreatic cells are not as easy to dissociate and aremarginally proliferative to nonproliferative in culture. Thus, thepreferred range for isolation of porcine pancreatic cells was determinedto be between about thirty-one (31) and about thirty-five (35) days ofdevelopment. This corresponds to fetal crown-to-rump (CRL) length ofbetween 25 and 45 mm.

Pancreatic cells within the preferred embryonic age range have some orall of the following characteristics: the cells form a monolayer ofadherent cells (i.e., they adhere to culture substrate, e.g., culturedish, forming fibroblast-like cells) when subconfluent; the cells (as asubconfluent monolayer of cells) are uniform in morphology, e.g., thereare few if any contaminating cells (e.g., cells that are associated withduct formation or cells that do not secrete or do not develop into cellsthat secrete pancreatic hormones or enzymes) and stain positive forglucagon and somatostatin but not for insulin; the cells are capable ofproliferating for an extended period of time under appropriateconditions, e.g., several months (six or more), in a growth medium andthe cells are maintained subconfluent; when the cells are allowed toreach confluence, they begin to form pseudo islet-like cell aggregatesspontaneously and stain positive for insulin, glucagon, andsomatostatin. Prior to the formation of islet-like aggregates, there isno detectable insulin staining. See FIG. 1A. The formation of islet-likeaggregates is necessary for the expression of insulin. See FIG. 1B.

Accordingly, this invention also features a population or a group of twoor more cells, of non-insulin-secreting porcine pancreatic cells havingthe ability to differentiate into insulin-secreting cells uponintroduction into a xenogeneic subject. The cells of the population aretypically obtained from a selected area of the developing gut, e.g., theprimordial pancreas, which is typically identified as an unlobulatedsolid tissue located around the duodenal loop just below the stomach.

The cells of the invention can be grown as a cell culture, i.e., as apopulation of cells which grow in vitro, in a medium suitable to supportthe growth of the cells. The characteristics of the cells when grown ascell cultures are described herein in detail. Media which can be used tosupport the growth of porcine pancreatic cells include mammalian cellculture media, such as those produced by Gibco BRL (Gaithersburg, Md.).See 1994 Gibco BRL Catalogue & Reference Guide. The medium can beserum-free but is preferably supplemented with animal serum such asfetal calf serum. A preferred medium is RPMI-1640 supplemented withfetal calf serum. The medium can be further supplemented with theembryonic proliferating agents described herein to induce or promoteproliferation of the porcine pancreatic cells.

As common methods of administering pancreatic cells to subjects,particularly human subjects, which are described in detail herein,include injection or implantation of the cells into target sites in thesubjects, the cells of the invention can be inserted into a deliverydevice which facilitates introduction by, injection or implantation, ofthe cells into the subjects. Such delivery devices include tubes, e.g.,catheters, for injecting cells and fluids into the body of a recipientsubject. In a preferred embodiment, the tubes additionally have aneedle, e.g., a syringe, through which the cells of the invention can beintroduced into the subject at a desired location. The porcinepancreatic cells of the invention can be inserted into such a deliverydevice, e.g., a syringe, in different forms. For example, the cells canbe suspended in a solution or embedded in a support matrix whencontained in such a delivery device. As used herein, the term "solution"includes a pharmaceutically acceptable carrier or diluent in which thecells of the invention remain viable. Pharmaceutically acceptablecarriers and diluents include saline, aqueous buffer solutions, solventsand/or dispersion media. The use of such carriers and diluents is wellknown in the art. The solution is preferably sterile and fluid to theextent that easy syringability exists. Preferably, the solution isstable under the conditions of manufacture and storage and preservedagainst the contaminating action of microorganisms such as bacteria andfungi through the use of, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. Solutions of the invention canbe prepared by incorporating porcine pancreatic cells as describedherein in a pharmaceutically acceptable carrier or diluent and, asrequired, other ingredients enumerated above, followed by filteredsterilization.

Support matrices in which the porcine pancreatic cells can beincorporated or embedded include matrices which are recipient-compatibleand which degrade into products which are not harmful to the recipient.Natural and/or synthetic biodegradable matrices are examples of suchmatrices. Natural biodegradable matrices include plasma clots, e.g.,derived from a mammal, and collagen matrices. Synthetic biodegradablematrices include synthetic polymers such as polyanhydrides,polyorthoesters, and polylactic acid. Other examples of syntheticpolymers and methods of incorporating or embedding cells into thesematrices are known in the art. See e.g., U.S. Pat. No. 4,298,002 andU.S. Pat. No. 5,308,701. These matrices provide support and protectionfor the fragile pancreatic cells in vivo and are, therefore, thepreferred form in which the pancreatic cells are introduced into therecipient subjects.

B. Modified Porcine Pancreatic Cells and Isolated Populations ofModified Porcine Pancreatic Cells

A further aspect of the invention is a porcine pancreatic cell which, inunmodified form, has at least one antigen on the cell surface which iscapable of stimulating an immune response against the cell in axenogeneic subject. To inhibit rejection of the cell when introducedinto the xenogeneic subject, the antigen on the cell surface is alteredprior to transplantation. In an unaltered state, the antigen on the cellsurface stimulates an immune response against the cell when the cell isadministered to a subject. By altering the antigen, the normalimmunological recognition of the porcine pancreatic cell by the immunesystem cells of the recipient is disrupted and additionally, "abnormal"immunological recognition of this altered form of the antigen can leadto porcine pancreatic cell-specific long term unresponsiveness in therecipient. It is likely that alteration of an antigen on the porcinepancreatic cell prior to introducing the cell into a subject interfereswith the initial phase of recognition of the porcine pancreatic cell bythe cells of the host's immune system subsequent to administration ofthe cell. Furthermore, alteration of the antigen can induceimmunological nonresponsiveness or tolerance, thereby preventing theinduction of the effector phases of an immune response (e.g., cytotoxicT cell generation, antibody production etc.) which are ultimatelyresponsible for rejection of foreign cells in a normal immune response.As used herein, the term "altered" encompasses changes that are made toat least one porcine pancreatic cell antigen(s) which reduce theimmunogenicity of the antigen to thereby interfere with immunologicalrecognition of the antigen(s) by the recipient's immune system. Anexample of an alteration of a porcine pancreatic cell antigen is bindingof a second molecule to the antigen. The second molecule can decrease orprevent recognition of the antigen as a foreign antigen by the recipientsubject's immune system.

Antigens to be altered according to the current invention includeantigens on a porcine pancreatic cell which can interact with an immunecell in a xenogeneic (or allogeneic) recipient subject and therebystimulate a specific immune response against the porcine pancreatic cellin the recipient. The interaction between the antigen and the immunecell can be an indirect interaction (e.g., mediated by soluble factorswhich induce a response in the immune cell, e.g., humoral mediated) or,preferably, is a direct interaction between the antigen and a moleculepresent on the surface of the immune cell (i.e., cell-cell mediated). Asused herein, the term "immune cell" is intended to include Tlymphocytes, B lymphocytes, monocytes and other antigen presentingcells. In a preferred embodiment, the antigen is one which interactswith a T lymphocyte in the recipient (e.g., the antigen normally bindsto a receptor on the surface of a T lymphocyte).

In one embodiment, the antigen on the porcine pancreatic cell to bealtered is an MHC class I antigen. Alternatively, an adhesion moleculeon the cell surface, such as ICAM-1, can be altered. An antigen whichstimulates a cellular immune response against the cell, such as an MHCclass I antigen, can be altered prior to transplantation by contactingthe cell with a molecule which binds to the antigen. A preferredmolecule for binding to the antigen is an antibody, or fragment thereof(e.g., an MHC class I antibody, or fragment thereof). A preferredantibody fragment is an F(ab')₂ fragment. Polyclonal or, morepreferably, monoclonal antibodies can be used. Other molecules which canbe used to alter an antigen (e.g., an MHC class I antigen) includepeptides and small organic molecules which bind to the antigen.Furthermore, two or more different epitopes on the same or differentantigens on the cell surface can be altered. A particularly preferredmonoclonal antibody for alteration of MHC class I antigens on porcinepancreatic cells is PT85 (commercially available from VeterinaryMedicine Research Development, Pullman Wash.). PT85 can be used alone toalter MHC class I antigens or, if each antibody is specific for adifferent epitope, PT85 can be used in combination with another antibodyknown to bind MHC class I antigens to alter the antigens on the cellsurface. Suitable methods for altering a surface antigen on a cell fortransplantation are described in greater detail in Faustman and Coe(1991) Science 252: 1700-1702 and PCT publication WO 92/04033. Methodsfor altering multiple epitopes on a surface antigen on a cell fortransplantation are described in greater detail in U.S. patentapplication Ser. No. 08/220,741, filed Mar. 31, 1994, now abandoned, thecontents of which are incorporated herein by reference. The altered(also referred to herein as "modified") porcine cells can comprise anisolated population of cells. The characteristics of such populationsare described above. The pancreatic cells to be modified can be obtainedfrom donor swine at the gestational ages described herein. Preferably,the modifications described herein are performed on porcine pancreaticcells prior to formation of islet-like aggregates or clusters. Preferreddonor swine are those which are essentially pathogen-free as describedherein.

C. Porcine Pancreatic Cells and Isolated Populations of PorcinePancreatic Cells Obtained from Essentially Patbogen-Free Swine

The invention also features a porcine pancreatic cell obtained from aswine which is essentially free from organisms or substances which arecapable of transmitting infection or disease to a xenogeneic recipient,e.g., a human recipient, of the cells. Typically, porcine pancreaticcells are obtained from a swine which is essentially free from pathogenswhich affect humans. For example, the pathogens from which the swine arefree include, but are not limited to, one or more of pathogens from thefollowing categories of pathogens: parasites, bacteria, mycoplasma, andviruses. The swine can be free from, for example, parasites such astoxoplasma and eperytherozoon, or mycoplasma, such as M. hyopneumonia.Examples of bacteria from which the swine can be free include brucella,listeria, mycobacterium TB, leptospirillum, and haemophilus suis.Additionally, the swine can be free from viruses such as zoonoticviruses (viruses which can be transferred from pigs to man under naturalconditions), viruses that can cross the placenta in pregnant sows, andneurotrophic viruses. Zoonotic viruses include, for example, a virus inthe rabies virus group, a herpes-like virus which causes pseudorabies,encephalomyocarditus virus, swine influenza Type A, transmissiblegastroenteritus virus, parainfluenza virus 3 and vesicular stomatitisvirus. Viruses that can cross the placenta include, for example, virusesthat cause porcine respiratory reproductive syndrome, a virus in therabies virus group, a herpes-like virus which causes pseudorabies,parvovirus, a virus that causes swine vesicular disease, techen (porcinepolio virus), hemmagluhnating encephalomyocarditus, cytomegalovirus,suipoxvirus, and swine influenza type A. Neurotrophic viruses include,for example, a virus in the rabies virus group, a herpes-like viruswhich causes pseudorabies, parvovirus, encephalomyocarditus virus, avirus which causes swine vesicular disease, porcine poliovirus (techen),a virus which causes hemmaglutinating encephalomyocarditus, adenovirus,parainfluenza 3 virus. Specific examples of viruses from which the swineare free include: a virus which causes (or results in) porcinerespiratory reproductive syndrome, a virus in the rabies virus group, aherpes-like virus which causes pseudorabies, parvovirus,encephalomyocarditus virus, a virus which causes swine vesiculardisease, porcine poliovirus (techen), a virus which causeshemmaglutinating encephalomyocarditus, cytomegalovirus, suipoxvirus,swine influenza type A, adenovirus, transmissible gastroenteritus virus,a virus which causes bovine viral diarrhea, parainfluenza virus 3, andvesicular stomatitis virus.

In one embodiment, the pigs from which pancreatic cells are isolated areessentially free from the following organisms: Toxoplasma,eperythrozoon, brucella, listeria, mycobacterium TB, leptospirillum,haemophilus suis, M. Hyopneumonia, a virus which causes porcinerespiratory reproductive syndrome, a virus which causes rabies, a viruswhich causes pseudorabies, parvovirus, encephalomyocarditus virus, avirus which causes swine vesicular disease, porcine polio virus(techen), a virus which causes hemagglutinating encephalomyocarditus,suipoxvirus, swine influenza type A, adenovirus, transmissiblegastroenteritis virus, a virus which causes bovine viral diarrhea, andvesicular stomatitis virus. The phrase "essentially free from organismsor substances which are capable of transmitting infection or disease toa xenogeneic recipient" (also referred to herein as "essentiallypathogen-free") when referring to a swine from which cells are isolatedmeans that swine does not contain organisms or substances in an amountwhich transmits infection or disease to a xenogeneic recipient, e.g. ahuman. Example III provides representative, but not limiting examples ofmethods for selecting swine which are essentially free from variouspathogens. The pancreatic cells of the invention can be isolated fromembryonic or post-natal swine which are determined to be essentiallyfree of such organisms. These swine are maintained under suitableconditions until used as a source of pancreatic cells.

Preferred gestational ages of the swine from which these cells areobtained are described in detail herein. Porcine pancreatic cellsobtained from essentially pathogen-free swine can additionally bemodified to reduce the immunogenicity of the cells followingadministration to a xenogeneic subject as described herein.

II. Methods of the Invention

A. Methods of Isolating Porcine Pancreatic Cells from Embryonic Swine

Other aspects of the invention include methods of isolating porcinepancreatic cells suitable for administration to a xenogeneic subject.These methods typically include isolating porcine pancreatic cells froma swine, e.g., an embryonic swine between about day thirty-one (31) andday thirty-five (35) of gestation, and optionally contacting the porcinepancreatic cells with at least one embryonic proliferating agent whichpromotes or induces cell proliferation in vitro or in vivo prior tointroduction of the cells into a subject. Porcine pancreatic cellsisolated according to the methods of the invention can be furthermodified as described herein for introduction into a xenogeneic subject.

Methods of isolating pancreatic cells from primordial gut tissue areknown in the art. For example, solid pancreatic tissue samples can bedissected from surrounding gut tissue, e.g., by dissecting the tissueunder a dissecting microscope. The cells in the pancreatic tissue samplecan then dissociated by mechanical means, e.g., chopping and/orsuccessive pipette trituration, or by chemical means, e.g., by use ofenzymes, such as trypsin or collagenase. The swine which are employed inthe method of the invention as a source of pancreatic cells includeembryonic swine (swine fetuses), postnatal swine, pathogen-freeembryonic swine, and pathogen-free postnatal swine. If an embryonicpathogen-free swine is to be used as a source of pancreatic cells, semenfrom a boar which has been tested to be essentially free ofdisease-transmitting organisms is employed to artificially inseminate afemale swine which is essentially free from such organisms. At aboutthirty-one (31) to about thirty-five (35) days of gestation, ahysterectomy is performed under appropriate conditions of sterility andthe fetuses are thereafter removed in their individual amniotic sacs.Appropriate pancreatic cells or tissue are thereafter recovered, asdescribed, for example, in Example I herein, under appropriateconditions of sterility.

The methods of isolating porcine pancreatic cells suitable foradministration to a xenogeneic subject can, optionally, further includeone or more of the following steps: administering the porcine pancreaticcells to a xenogeneic subject prior to formation of insulin-secretingislet-like aggregates or clusters in culture; administering the porcinepancreatic cells to a xenogeneic subject after the cells forminsulin-secreting islet-like aggregates or clusters in culture; andadministering the porcine pancreatic cells to a xenogeneic subject asnon-insulin-secreting cells which are capable of differentiating in vivoto form insulin-secreting cells. The in vitro and in vivocharacteristics of the porcine pancreatic cells of the invention aredescribed in further detail herein.

B. Methods of Promoting or Inducing Proliferation of Porcine PancreaticCells

Further aspects of the invention include methods of promoting orinducing proliferation of embryonic porcine pancreatic cells. Thesemethods include contacting embryonic porcine pancreatic cells, in vitroor in vivo, with at least one embryonic proliferating agent, whichpromote(s) or induce(s) proliferation of the cells. The phrase"embryonic proliferating agent" is intended to include agents whichpromote or enhance the proliferation of embryonic porcine pancreaticcells. Thus, embryonic proliferating agents which promote or induceproliferation of the porcine pancreatic cells include substances whichincrease the number of times that an embryonic porcine pancreatic cellmultiplies or reproduces to form two cells (e.g., doubling time) in agiven period of time. Specific examples of growth factors include PDGFand EGF and their equivalents. When the porcine pancreatic cells of theinvention are contacted with both PDGF and EGF, their doubling time canbe decreased by about 30-50% (e.g., 80 hours without growth factors vs.58 hours with growth factors). It should be understood that the phrase"embryonic proliferating agent" also includes growth factors for whichembryonic porcine pancreatic cells of a certain gestational age (e.g.,between about 31 to about 35 days) express a receptor. The methods ofthe invention allow the efficient production of large numbers of porcinepancreatic cells for introduction into xenogeneic subjects. This is animportant as about ten to fifty to about one hundred fifty millionporcine pancreatic cells are required to treat one human having adisease characterized by insufficient insulin activity. According to themethods of this invention, one fetal pig yields about one and a halfmillion pancreatic cells. Thus, about ten doublings (about 24 days) ofthese fetal pancreatic cells results in a number of cells sufficient forintroduction into a human subject. If several fetal pigs, e.g., a litterof fetal pigs, are used as donors of pancreatic cells, sufficientnumbers of cells can be generated for introduction into a humanrecipient in a matter of days.

C. Methods of Treating Diseases Characterized by Insufficient InsulinActivity Using Porcine Pancreatic Cells

Still further aspects of the invention include methods of treatingdiseases characterized by insufficient insulin activity in a subject,particularly a human subject. These methods include administering to axenogeneic subject, an isolated population of non-insulin-secretingporcine pancreatic cells having the ability to differentiate to forminsulin-secreting cells after administration to the subject. Suchpopulations of cells are described in detail herein. The terms"introduction", "administration", and "transplantation" are usedinterchangeably herein and refer to delivery of cells to a xenogeneicsubject by a method or route which delivers the cells to a desiredlocation. The term "treating" as used herein includes reducing oralleviating at least one adverse effect or symptom, e.g, absolute orrelative insulin deficiency, fasting hyperglycemia, glycosuria,development of atherosclerosis, microangiopathy, nephropathy, andneuropathy, of diseases characterized by insufficient insulin activity.As used herein, the language "diseases characterized by insufficientinsulin activity" include diseases in which there is an abnormalutilization of glucose due to abnormal insulin function. Abnormalinsulin function includes any abnormality or impairment in insulinproduction, e.g., expression and/or transport through cellularorganelles, such as insulin deficiency resulting from, for example, lossof β cells as in IDDM (Type I diabetes), secretion, such as impairmentof insulin secretory responses as in NIDDM (Type II diabetes), form ofthe insulin molecule itself, e.g., primary, secondary or tertiarystructure, effects of insulin on target cells, e.g., insulin-resistancein bodily tissues, e.g., peripheral tissues, and responses of targetcells to insulin. See Braunwald, E. et al. eds. Harrison's Principles ofInternal Medicine, Eleventh Edition (McGraw-Hill Book Company, New York,1987) pp. 1778-1797; Robbins, S. L. et al. Pathologic Basis of Disease,3rd Edition (W. B. Saunders Company, Philadelphia, 1984) p. 972 forfurther descriptions of abnormal insulin activity in IDDM and NIDDM andother forms of diabetes.

The porcine pancreatic cells are administered to the subject by anyappropriate route which results in delivery of the cells to a desiredlocation in the subject where the cells can proliferate and secrete apancreatic hormone, e.g., insulin, or enzyme. Preferred locations forpancreatic cell administration include those which rapidly vascularize.Common methods of administering pancreatic cells to subjects,particularly human subjects, include implantation of cells in a pouch ofontentum (Yoneda, K. et al. (1989) Diabetes 38 (Suppl. 1): 213-216),intraperitoneal injection of the cells, (Wahoff, D. C. et al. (1994)Transplant. Proc.26: 804), implantation of the cells under the kidneycapsule of the subject (See, e.g., Liu, X. et al. (1991) Diabetes 40:858-866; Korsgren, O. et al. (1988) Transplantation 45(3): 509-514;Simeonovic, D. J. et al. (1982) Aust. J. Exp. Biol. Med. Sci. 60: 383),and intravenous injection of the cells into, for example, the portalvein (Braesch, M. K. et al. (1992) Transplant. Proc. 24(2): 679-680;Groth, C. G. et al. (1992) Transplant. Proc. 24(3): 972-973). Tofacilitate transplantation of the pancreatic cells under the kidneycapsule, the cells can be embedded in a plasma clot prepared from, e.g.,plasma from the recipient subject (Simeonovic, D. J. et al. (1982) Aust.J. Exp. Biol. Med. Sci. 60: 383) or a collagen matrix. Cells can beadministered in a pharmaceutically acceptable carrier or diluent asdescribed herein.

This invention further pertains to methods of treating diseasescharacterized by insufficient insulin activity in a subject,particularly a human subject, in which an isolated population of porcinepancreatic cells obtained from an embryonic pig between about day 31 andday 35 of gestation, which form insulin-secreting islet-like aggregatesor clusters in culture, and which secrete insulin after administrationto the subject is administered to the subject. As described herein,pancreatic cells obtained from embryonic pigs between about day 31 and35 can be cultured as a monolayer of adherent non-insulin secretingcells. When these cells are allowed to reach confluence, they formislet-like aggregates or clusters and begin to secrete pancreatichormones, such as insulin, glucagon, and somatostatin, and enzymes. Atthis point, such aggregates can be isolated, pooled, and administered toa recipient subject wherein they secrete insulin. About 100,000 to500,000 aggregates, each of which contains about 300 to 500 cells, canbe used to treat one human. This number of cells can be generated fromone pig fetus after about ten doublings (about 24 days) or from a litter(6-10) of fetal pigs after only a few days of doubling (about 2-10days). Additional porcine pancreatic cells and isolated populations ofsuch cells which can be administered to a xenogeneic subject accordingto this method include embryonic porcine pancreatic cells, embryonicporcine pancreatic cells obtained from an essentially pathogen-free pig,modified porcine pancreatic cells, modified porcine pancreatic cellsobtained from an essentially pathogen-free pig, modified embryonicporcine pancreatic cells, and modified embryonic porcine pancreaticcells obtained from an essentially pathogen-free pig. These and otherporcine pancreatic cells are described in detail herein.

The porcine pancreatic cells of the invention can be administered to axenogeneic subject having a disease characterized by insufficientinsulin activity in combination with administration of an agent whichinhibits T cell activity in the subject. As used herein, an agent whichinhibits T cell activity is defined as an agent which results in removal(e.g., sequestration) or destruction of T cells within a subject orinhibits T cell functions within the subject (i.e., T cells may still bepresent in the subject but are in a non-functional state, such that theyare unable to proliferate or elicit or perform effector functions, e.g.cytokine production, cytotoxicity etc.). The term "T cell" encompassesmature peripheral blood T lymphocytes. The agent which inhibits T cellactivity may also inhibit the activity or maturation of immature T cells(e.g., thymocytes).

A preferred agent for use in inhibiting T cell activity in a recipientsubject is an immunosuppressive drug. The term "immunosuppressive drugor agent" is intended to include pharmaceutical agents which inhibit orinterfere with normal immune function. A preferred immunsuppressive drugis cyclosporin A. Other immunosuppressive drugs which can be usedinclude FK506, and RS-61443. In one embodiment, the immunosuppressivedrug is administered in conjunction with at least one other therapeuticagent. Additional therapeutic agents which can be administered includesteroids (e.g., glucocorticoids such as prednisone, methyl prednisoloneand dexamethasone) and chemotherapeutic agents (e.g., azathioprine andcyclosphosphamide). In another embodiment, an immunosuppressive drug isadministered in conjunction with both asteroid and a chemotherapeuticagent. Suitable immunosuppressive drugs are commercially available(e.g., cyclosporin A is available from Sandoz, Corp., East Hanover,N.J.).

An immunsuppressive drug is administered in a formulation which iscompatible with the route of administration. Suitable routes ofadministration include intravenous injection (either as a singleinfusion, multiple infusions or as an intravenous drip over time),intraperitoneal injection, intramuscular injection and oraladministration. For intravenous injection, the drug can be dissolved ina physiologically acceptable carrier or diluent (e.g., a buffered salinesolution) which is sterile and allows for syringability. Dispersions ofdrugs can also be prepared in glycerol, liquid polyethylene glycols, andmixtures thereof and in oils. Convenient routes of administration andcarriers for immunsuppressive drugs are known in the art. For example,cyclosporin A can be administered intravenously in a saline solution, ororally, intraperitoneally or intramuscularly in olive oil or othersuitable carrier or diluent.

An immunosuppressive drug is administered to a recipient subject at adosage sufficient to achieve the desired therapeutic effect (e.g.,inhibition of rejection of transplanted cells). Dosage ranges forimmunosuppressive drugs, and other agents which can be coadministeredtherewith (e.g., steroids and chemotherapeutic agents), are known in theart (See e.g., Freed et al. New Engl. J. Med. (1992) 327: 1549: Spenceret al. (1992) New Engl. J. Med. 327: 1541; Widner et al. (1992) NewEngl. J. Med. 327: 1556; Lindvall et al. (1992) Ann. Neurol. 31: 155;and Lindvall et al. (1992) Arch. Neurol. 46: 615). A preferred dosagerange for immunosuppressive drugs, suitable for treatment of humans, isabout 1-30 mg/kg of body weight per day. A preferred dosage range forcyclosporin A is about 1-10 mg/kg of body weight per day, morepreferably about 1-5 mg/kg of body weight per day. Dosages can beadjusted to maintain an optimal level of the immunosuppressive drug inthe serum of the recipient subject. For example, dosages can be adjustedto maintain a preferred serum level for cyclosporin A in a human subjectof about 100-200 ng/ml. It is to be noted that dosage values may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual. Dosage regimens may be adjusted over time to provide theoptimum therapeutic response according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that the dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed composition.

In one embodiment of the invention, an immunsuppressive drug isadministered to a subject transiently for a sufficient time to inducetolerance to the transplanted cells in the subject. Transientadministration of an immunosuppressive drug has been found to inducelong-term graft-specific tolerance in a graft recipient (See Brunson etal. (1991) Transplantation 52: 545; Hutchinson et al. (1981)Transplantation 32: 210; Green et al. (1979) Lancet 2: 123; Hall et al.(1985) J. Exp. Med 162: 1683). Administration of the drug to the subjectcan begin prior to transplantation of the cells into the subject. Forexample, initiation of drug administration can be a few days (e.g., oneto three days) before transplantation. Alternatively, drugadministration can begin the day of transplantation or a few days(generally not more than three days) after transplantation.Administration of the drug is continued for sufficient time to inducedonor cell-specific tolerance in the recipient such that donor cellswill continue to be accepted by the recipient when drug administrationceases. For example, the drug can be administered for as short as threedays or as long as three months following transplantation. Typically,the drug is administered for at least one week but not more than onemonth following transplantation. Induction of tolerance to thetransplanted cells in a subject is indicated by the continued acceptanceof the transplanted cells after administration of the immunosuppressivedrug has ceased. Acceptance of transplanted tissue can be determinedmorphologically (e.g., with skin grafts by examining the transplantedtissue or by biopsy) or by assessment of the functional activity of thegraft.

Another type of agent which can be used to inhibit T cell activity in asubject is an antibody, or fragment or derivative thereof, whichdepletes or sequesters T cells in a recipient. Antibodies which arecapable of depleting or sequestering T cells in vivo when administeredto a subject are known in the art. Typically, these antibodies bind toan antigen on the surface of a T cell. Polyclonal antisera can be used,for example anti-lymphocyte serum. Alternatively, one or more monoclonalantibodies can be used. Preferred T cell-depleting antibodies includemonoclonal antibodies which bind to CD2, CD3, CD4 or CD8 on the surfaceof T cells. Antibodies which bind to these antigens are known in the artand are commercially available (e.g., from American Type CultureCollection). A preferred monoclonal antibody for binding to CD3 on humanT cells is OKT3 (ATCC CRL 8001). The binding of an antibody to surfaceantigens on a T cell can facilitate sequestration of T cells in asubject and/or destruction of T cells in a subject by endogenousmechanisms. Alternatively, a T cell-depleting antibody which binds to anantigen on a T cell surface can be conjugated to a toxin (e.g., ricin)or other cytotoxic molecule (e.g., a radioactive isotope) to facilitatedestruction oft cells upon binding of the antibody to the T cells. SeeU.S. patent application Ser. No.: 08/220,724, filed Mar. 31, 1994, forfurther details concerning the generation of antibodies which can beused in the present invention.

Another type of antibody which can be used to inhibit T cell activity ina recipient subject is an antibody which inhibits T cell proliferation.For example, an antibody directed against a T cell growth factor, suchas IL-2, or a T cell growth factor receptor, such as the IL-2 receptor,can inhibit proliferation of T cells (See e.g., DeSilva, D. R. et al.(1991) J. Immunol. 147: 3261-3267). Accordingly, an IL-2 or an IL-2receptor antibody can be administered to a recipient to inhibitrejection of a transplanted cell (see e.g. Wood et al. (1992)Neuroscience 49: 410). Additionally, both an IL-2 and an IL-2 receptorantibody can be coadministered to inhibit T cell activity or can beadministered with another antibody (e.g., which binds to a surfaceantigen on T cells).

An antibody which depletes, sequesters or inhibits T cells within arecipient can be administered at a dose and for an appropriate time toinhibit rejection of cells upon transplantation. Antibodies arepreferably administered intravenously in a pharmaceutically acceptablecarrier or diluent (e.g., a sterile saline solution). Antibodyadministration can begin prior to transplantation (e.g., one to fivedays prior to transplantation) and can continue on a daily basis aftertransplantation to achieve the desired effect (e.g., up to fourteen daysafter transplantation). A preferred dosage range for administration ofan antibody to a human subject is about 0.1-0.3 mg/kg of body weight perday. Alternatively, a single high dose of antibody (e.g., a bolus at adosage of about 10 mg/kg of body weight) can be administered to a humansubject on the day of introduction of the pancreatic cells into thesubject. The effectiveness of antibody treatment in depleting T cellsfrom the peripheral blood can be determined by comparing T cell countsin blood samples taken from the subject before and after antibodytreatment. Dosage regimes can be adjusted over time to provide theoptimum therapeutic response according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions. Dosage ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition.

To assess their therapeutic potential, the porcine pancreatic cells ofthe invention can be introduced into existing animal models fordiabetes. These models include, for example, mice in which diabetes isinduced by, for example, intravenous injection of alloxan (Korsgren, O.et al. (1993) Surgery 113: 205-214) or administration of streptozocin(Liu, X et al. (1991) Diabetes 858-866). Other animal models of diabetesinclude db/db and ob/ob mouse lines (Pittman et al. (1994) Transplant.Proc. 26: 1135-1137). Therapeutic efficacy in these models of diabetescan be predictive of therapeutic efficacy in humans. Groth, C. G. et al.(1992) Transplant. Proc. 24(3): 972-973. The therapeutic efficacy of theadministered porcine pancreatic cells is typically determined by, forexample, measurement of blood glucose concentrations using, for example,an intravenous glucose tolerance test, before and after administrationof the porcine pancreatic cells. Normalization of hyperglycemiademonstrates that the administered porcine pancreatic cells can be usedto treat diseases characterized by insufficient insulin secretion. Othermethods of determining the therapeutic potential are histologicalexamination of the pancreatic cell graft (via a biopsy), e.g., bystaining for insulin, and measurement of insulin levels in blood by, forexample, radioimmunoassay.

This invention is further illustrated by the following examples which inno way should be construed as being further limiting. The contents ofall cited references (including literature references, issued patents,published patent applications, and co-pending patent applications) citedthroughout this application are hereby expressly incorporated byreference.

EXAMPLES Example I Dissection of Pancreatic Cells from Embryonic Swine

Female pigs were inseminated 31-35 days prior to euthanization andremoval of uterus. After uterus was surgically removed and transportedto sterile laboratory facilities, fetuses were delivered into a steriledish containing calcium-magnesium free phosphate buffered saline (PBS)in a horizontal laminar flow hood. Fetuses at this stage of developmentranged from 25-45 mm in length (crown-to-rump length).

Fetuses were removed from the storage dish containing PBS and placed ina sterile dissecting dish containing PBS. The dissecting dish was thenplaced on the stage of a dissecting microscope. With the aid of thedissecting microscope, a longitudinal incision was made down the midlineof the fetus. Next, an incision was made at right angles to the firstincision just below the ribs and chest cavity. The overlying skin waspulled back to expose the internal organs of the fetus. The liver, whichis the large red organ occupying virtually the entire ventral bodycavity, was located and removed. After the liver was removed the stomachand small intestine were located. The stomach was white and kidneyshaped at this stage of development. Once located, the stomach wasfollowed to the point where it joins the small intestine. At thejuncture between the stomach and the small intestine, the dorsal aspectof the fetal pancreas can be identified lying in close apposition to thestomach. The fetal pancreas was the same color as the stomach anddifficult to locate. Once the pancreas had been located, it was observedthat the pancreas is a small tongue-like structure that was U-shaped andsurrounded the posterior half of the stomach. With the fetal pancreaslocated, it was dissected away from the stomach with a pair of forcepsand micro-scissors then placed in a tube containing sterile PBS. Theprocedure was repeated for all fetuses from a single donor sow.

The PBS was removed from the tube containing dissected pancreases. Thetissue was then resuspended in 1.5 ml of 0.05% Trypsin, 0.53 mM EDTA andincubated at 37° C. for 15 minutes. Tissue was dissociated bytriturating with a pasteur pipette until a uniform cell suspension wasformed. Trypsin was stopped by adding 5 ml of medium (RPMI-1640+10%FCS), then the cells were collected at 1000 RPM for 5 minutes at 25° C.Cells were resuspended in culture media (RPMI-1640+10% FCS+5 ng/mlPDGF+100 ng/ml EGF) and plated in sterile tissue culture dishes. Cellswere then allowed to adhere and grow at 37° C. in an incubator with 5%CO₂. FIGS. 1 A-1B depict insulin staining of the cultured fetal pigpancreatic cells. FIG. 1A is a phase micrograph of fetal pig pancreaticcells in monolayer prior to formation of islet-like clusters after twoweeks of culture. No insulin staining can be detected in these cells atthis time. FIG. 1B is a phase micrograph of fetal pig pancreatic cellsafter three weeks of culture that were allowed to form islet-likeclusters (indicated by arrows). These cells stained positive forinsulin.

The growth rate behavior of the pancreatic cells includes a doublingtime of 80 hours without growth factors and 58 hours with growthfactors. Fourteen fetuses provided approximately 1-2×10⁶ pancreaticcells. The cells can be cultured for 30 days to yield 4-8×10⁹ cells. Thelength of time required for islet-like aggregate or cluster formation is4-7 days after the cells reach confluence.

Example II Introduction of Porcine Pancreatic Cells into XenogeneicRecipients and Demonstration of Insulin-Secretion In Vivo

All transplantations were done into nude mice recipients. Mice wereanaesthetized by intraperitoneal administration of Avertin (250 mg/kgbody weight) and a flank incision was made to expose the kidney. A smallincision was made on the kidney and a small fire polished glass rod wasinserted between the kidney epithelium and the kidney tissue to create aspace for cells to be transplanted. Prior to transplantation, 10⁶proliferating cells (which did not stain for insulin) were eitherimmobilized in a rat tail collagen matrix or in a blood clot. Forimmobilization in rat tail collagen, 8 parts collagen at 5.29 mg/ml+1part 0.1M NaOH+1 part 10×Hank's balanced salt solution (HBSS) werecombined and the pH was adjusted to 7.4 with 0.1M NaOH. Collagen waskept at 4° C. until addition to cells. Collagen did not readily gel at4° C. or when pH was below 7.4, therefore, adjusting pH and warming toroom temperature were essential for formation of a collagen gel.Immobilized cells were placed with forceps through the incision made onthe kidney in the space previously created. The skin incision was thenclosed with a wound clip and the animal transferred to a cage forrecovery. In the first experiments, 12 animals were transplanted: 6animals received cells immobilized in collagen. At one monthpost-transplant, 3 animals from each group were sacrificed and thetransplanted kidneys processed for histology and immunostaining. Theremaining animals are sacrificed at 3 months post-transplantation. Forthe animals that were sacrificed, grafts were found in 5 or 6 animals:one animal inadvertently received 1/10 the number of cells intended andthat was the animal with no graff. There was no apparent differencebetween grafts derived from cells immobilized in blood clots orcollagen. All grafts stained positive for insulin. For example, FIG. 2Ais a section from transplanted kidney showing the graft stained withaldehyde-fuchsin. Insulin-containing cells stain darkly and can be seenscattered throughout the graft. FIG. 2B shows insulin staining from thesame graft which is stained with a primary Mouse Insulin Ab monoclonal(Chemicon Intl., Inc., Temecula, Calif.) and a secondary goat-anti-mouseantibody conjugated with horseradish peroxidase (Vector Labs Inc.,Burlingame, Calif.). The insulin-containing cells within the graft stainmore darkly than the other cells. In both figures, the insulin-positivecells are indicated by arrows, the donor graft is marked (G), and therecipient mouse kidney is marked (K).

Example III Methods of Producing Essentially Pathogen-Free Swine fromwhich Pancreatic Cells of the Invention can be Obtained

A. Collecting, processing, and analyzing pig fecal samples for signs ofpathogens

Feces are extracted from the pig's rectum manually and placed in asterile container. About a 1.5 cm diameter portion of the specimen wasmixed thoroughly in 10 ml of 0.85% saline. The mixture is then strainedslowly through a wire mesh strainer into a 15 ml conical centrifuge tubeand centrifuged at 650×g for 2 minutes to sediment the remaining fecalmaterial. The supernatant is decanted carefully so as not to dislodgethe sediment. and 10% buffered formalin was added to the 9 ml mark,followed by thorough mixing. The mixture is allowed to stand for 5minutes. 4 ml of ethyl acetate is added to the mixture and the mixtureis capped and mixed vigorously in an inverted position for 30 seconds.The cap is then removed to allow for ventilation and then replaced. Themixture is centrifuged at 500×g for 1 minute (four layers should result:ethyl acetate, debris plug, formalin and sediment). The debris plug isrimmed using an applicator stick. The top three layers are carefullydiscarded by pouring them off into a solvent container. The debrisattached to the sides of the tube is removed using a cotton applicatorswab. The sediment is mixed in either a drop of formalin or the smallamount of formalin which remains in the tube after decanting. Twoseparate drops are placed on a slide to which a drop of Lugol's iodineis added. Both drops are coverslipped and carefully examined for signsof pathogens, e.g., protozoan cysts of trophozoites, helminth eggs andlarvae. Protozoan cyst identification is confirmed, when required, bytrichrome staining.

B. Co-cultivation assay for detecting the presence of human and animalviruses in pig cells

Materials

Cell Lines

African green monkey kidney, (VERO), cell line American Type CultureCollection, (ATCC CCL81), human embryonic lung fibroblasts, (MRC-5) cellline American Type Culture Collection, (ATCC CCL 171), porcine kidney,(PK-15), cell line American Type Culture Collection, (ATCC CRL 33),porcine fetal testis, (ST), cell line American Type Culture Collection,(ATCC CRL 1746).

Medium, Antibiotics, and Other Cells, and Equipment

Fetal calf serum, DMEM, Penicillin 10,000 units/ml, Streptomycin 10mg/ml, Gentamicin 50 mg/ml, guinea pig erythrocytes, chickenerythrocytes, porcine erythrocytes, Negative Control (sterile cellculture medium), Positive Controls: VERO and MRC-5 Cells: Poliovirustype 1 attenuated, (ATCC VR-1 92) and Measles virus, Edmonston strain,(ATCC VR-24), PK-15 and ST Cells: Swine influenza type A, (ATCC VR-99),Porcine Parvovirus, (ATCC VR-742), and Transmissible gastroenteritis ofswine, (ATCC VR-743). Equipment: tissue Culture Incubator, InvertedMicroscope, Biological Safety Cabinet.

These materials can be used in a co-cultivation assay (a process wherebya test article is inoculated into cell lines (VERO, MRC-5, PK1 5, andST) capable of detecting a broad range of human, porcine and otheranimal viruses). Hsuing, G. D., "Points to Consider in theCharacterization of Cell Lines Used to Produce Biologicals" inDiagnostic Virology, 1982 (Yale University Press, New Haven, Conn.,1982).

Experimental Design and Methodology

A total of three flasks (T25) of each cell line are inoculated with atleast 1 ml of test article. Three flasks of each cell line can also beinoculated with the appropriate sterile cell culture medium as anegative control. Positive control viruses are inoculated into threeflasks of each cell line. After an absorption period, the inoculate isremoved and all flasks incubated at 35°-37° C. for 21 days. All flasksare observed at least three times per week for the development ofcytopathic effects, (CPE), of viral origin. Harvests are made from anyflasks inoculated with the test article that show viral CPE.

At Day 7 an aliquot of supernatant and cells from the flasks of eachtest article are collected and at least 1 ml is inoculated into each ofthree new flasks of each cell line. These subcultures are incubated at35°-37° C. for at least 14 days. All flasks are observed and tested asdescribed above.

At Day 7, the flasks from each test article are also tested for viralhemadsorption, (HAd), using guinea pig, monkey and chicken erythrocytesat 2°-8° C. and 35°-37° C. at 14 days postinoculation.

At Day 21, if no CPE is noted, an aliquot of supematant from each flaskis collected, pooled, and tested for viral hemagglutination, (HA), usingguinea pig, monkey, and chicken erythrocytes at 2°-8° C. and 35°-37° C.Viral identification is based on characteristic viral cytopathic effects(CPE) and reactivity in HA testing.

The test samples are observed for viral cytopathic effects in thefollowing manner: All cultures are observed for viral CPE at least threetimes each week for a minimum of 21 days incubation. Cultures areremoved from the incubator and observed using an inverted microscopeusing at least 40X magnification. 100X or 200X magnification is used asappropriate. If any abnormalities in the cell monolayers, includingviral CPE, are noted or any test articles cause total destruction of thecell monolayer, supernatant and cells are collected from the flasks andsamples are subcultured in additional flasks of the same cell line.Samples can be stored at -60° to -80° C. until subcultured. After 7 and14 days incubation, two blind passages are made of each test article bycollecting supernatant and cells from all flasks inoculated with eachsample. Samples can be stored at -60° to -80° C. until subcultured.

Hemadsorbing viruses are detected by the following procedure: after 21days of incubation, a hemadsorption test is performed on the cells todetect the presence of hemadsorbing viruses. The cells are washed 1-2times with approximately 5 mls of PBS. One to two mls of the appropriateerythrocyte suspension (either guinea pig, porcine, or chickenerythrocytes), prepared as described below, is then added to each flask.The flasks are then incubated at 2°-8° C. for 15-20 minutes, after whichtime the unabsorbed erythrocytes are removed by shaking the flasks. Theerythrocytes are observed by placing the flasks on the lowered stage ofa lab microscope and viewing them under low power magnification. Anegative result is indicated by a lack of erythrocytes adhering to thecell monolayer. A positive result is indicated by the adsorption of theerythrocytes to the cell monolayer.

Hemagglutination testing, described in detail below, is also performedafter 21 days of incubation of the subcultures. Viral isolates areidentified based on the cell line where growth was noted, thecharacteristics of the viral CPE, the hemadsorption reaction, andhemagglutination reactions, as appropriate. The test article isconsidered negative for the presence of a viral agent, if any of thecell lines used in the study demonstrate viral, CPE, HA, or HAd in avalid assay.

C. Procedure for preparing and maintaining cell lines used to detectviruses in pig cells

Materials

Fetal calf serum (FCS), DMEM, Penicillin 10,000 unit/ml, Streptomycin 10mg/ml, Gentamicin 50 mg/ml, T25 tissue culture flasks, tissue cultureincubator (5% CO₂, 37° C.)

Procedure

Aseptic techniques are followed when performing inoculations andtransfers. All inoculations and transfers are performed in a biologicalsafety cabinet. Media is prepared by adding 10% FCS for initial seeding,5% FCS for maintenance of cultures, as well as 5.0 ml ofpenicillin/streptomycin and 0.5 ml of gentamicin per 500 ml media.Sufficient media is added to cover the bottom of a T25 tissue cultureflask. The flask is seeded with the desired cell line and incubated at37° C., 5% CO₂ until cells are 80 to 100% confluent. The flasks are theninoculated with virus (QCP25).

D. Preparation of erythrocyte (rbc) suspensions used in hem adsorption(HAd) and hemagglutination (HA) virus detection testing

Materials

Phosphate buffered saline, (PBS), pH 7.2, guinea pig erythrocytes stocksolution, porcine erythrocytes stock solution, chicken erythrocytesstock solution, sterile, disposable centrifuge tubes, 15 or 50 mlLaboratory centrifuge

Procedure

An appropriate amount of erythrocytes (rbc) is obtained from stocksolution. The erythrocytes are washed 3 times with PBS by centrifugationat approximately 1000×g for 10 minutes. A 10% suspension is prepared byadding 9 parts of PBS to each one part of packed erythrocytes. The 10%rcb suspensions are stored at 2°-8° C. for no more than one week. 0.5%ecb suspensions are prepared by adding 19 parts of PBS to each one partof 10% rbc suspension. Fresh 0 5% rbc suspensions are prepared prior toeach day's testing.

Hemagglutination (HA) Test

A hemagglutination test is a test that detects viruses with the propertyto agglutinate erythrocytes, such as swine influenza type A,parainfluenza, and encephalomyocarditus viruses, in the test article.Hsuing, G. D. (1982) Diagnostic Virology (Yale University Press, NewHaven, Conn.);. Stites, Daniel P and Terr, Abba I, (1991), Basic andClinical Immunology (Appleton & Lange, East Norwalk, Conn.).

Materials

Supernatants from flasks of the VERO cell line, MRC-5 inoculated withthe test article, flasks of positive and negative controls, phosphatebuffered saline (PBS), pH 7.2, guinea pig erythrocytes (GPRBC), 0.5%suspension in PBS, chicken erythrocytes (CRBC), 0.5% suspension in PBS,porcine erythrocytes (MRBC), 0.5% suspension in PBS

Procedure

All sample collection and testing is performed in an approved biologicalsafety cabinet. 0.5% suspensions of each type of erythrocytes areprepared as described above. The HA test on all cell lines inoculatedwith samples of the test articles at least 14 days post-inoculation.Positive and negative control cultures are included for each sample andmonolayers are examined to ensure that they are intact prior tocollecting samples.

At least 1 ml of culture fluid from each flask inoculated with the testarticle is collected and pooled. 1 ml samples from the negative andpositive control cultures are also collected and pooled. A set of tubesis labeled with the sample number and type of erythrocyte (distinguishpositive and negative suspension) to be added. Racks may be labeled todifferentiate the type of erythrocyte. 0.1 ml of sample is added to eachtube. 0.1 ml of the appropriate erythrocyte suspension is added to eachtube. Each tube is covered with parafilm and mixed thoroughly. One setof tubes is incubated at 2°-8° C. until tight buttons form in thenegative control in about 30-60 minutes. Another set of tubes isincubated at 35°-37° C. until tight buttons form in the negative controlin about 30-60 minutes.

Formation of a tight button of erythrocytes indicates a negative result.A coating of the bottom of the tube with the erythrocytes indicates apositive result.

E. Methods used for fluorescent antibody stain of cell suspensionsobtained from flasks used in detection of viruses in porcine cells usingcell culture techniques (as described in Sections B and C)

Materials

Pseudorabies, parvovirus, enterovirus, adenovirus, transmissibleGastroenteritis Virus. bovine viral diarrhea, encephalomyocarditusvirus, parainfluenza, vesicular stomatitis virus., microscope slides,PBS, incubator with humidifying chamber at 36° C., Evan's blue coutnerstain, DI Water, fluorescent microscope, trypsin, serum containingmedia, acetone, T25 Flask.

Procedure

Cells (described in Sections B and C) are trypsinized to detach themfrom the T25 flask and sufficient media is added to neutralize trypsinactivity. A drop of cell suspension is placed on each microscope slideand allowed to air dry. A slide for each fluorescent antibody isprepared. Cells are fixed by immersion in acetone for five minutes. Eachfluorescent antibody solution is placed on each slide to cover cells andthe slides are incubated in humidifying chamber in incubator at 36° C.for 30 minutes. The slides are then washed in PBS for five minutes. Thewash is repeated in fresh PBS for five minutes followed by a rinse withDI water.

The cells are counterstained by placing Evan's blue solution on eachslide to cover cells for five minutes at room temperature. The slidesare then washed in PBS for five minutes. The wash is repeated in freshPBS for five minutes followed by a rinse with DI water. The slides arethen allowed to air dry. Each slide is inspected under a fluorescentmicroscope. Any fluorescent inclusion bodies characteristic of infectionare considered a positive result for the presence of virus.

F. Procedures for Defining Bacteremic Pigs

Materials

Anaerobic BMB agar (5% sheep blood, vitamin K and hemin [BMB/blood]),chocolate Agar with Iso Vitalex, Sabaroud dextrose agar/Emmons, 70%isopropyl alcohol swabs, betadine solution, 5% CO₂ incubator at 35°-37°C., anaerobic blood agar plate, gram stain reagents (Columbia BrothMedia), aerobic blood culture media (anaerobic brain heart infusion withvitamin K& hemin), septicheck media system, vitek bacterialidentification system, laminar flow hood, microscope, and bacteroids andBacillus stocks

Procedure

Under a laminar flow hood, disinfect the tops of bottles for aerobic andanaerobic blood cultures of blood obtained from pig with 70% isopropylalcohol, then with betadine The rubber stopper and cap from the aerobicblood culture bottle are removed and a renal septicheck media system isattached to the bottle. The bottles are incubated in 5% CO₂ for 21 daysat 35°-37° C., and observed daily for any signs of bacterial growth(i.e. gas bubbles, turbidity, discoloration or discrete clumps).Negative controls consisting of 5 cc of sterile saline in each bottleand positive controls consisting of Bacillus subtills in the aerobicbottle and Bacteriodes Vulgaris in the anaerobic bottle are used. Ifsigns of bacterial growth are observed, a Gram stain is prepared andviewed microscopically at 100× oil immersion for the presence of anybacteria or fungi. The positive bottles are then subcultured onto bothchocolate agar plates with Iso Vitlex and onto BMB plates. The chocolateplate is incubated at 35°-37° C. in 5% CO₂ for 24 hours and the BMBanaerobically at 35°-37° C. for 48 hours. Any yeast or fungi that is inevidence at gram stain is subcultured onto a Sabaroud dextrose/Emmonsplate. The Vitek automated system is used to identify bacteria andyeast. Fungi are identified via their macroscopic and microscopiccharacteristic. If no signs of growth are observed at the end of 21days, gram stain is prepared and observed microscopically for thepresence of bacteria and fungi.

Absence of growth in the negative control bottles and presence of growthin the positive control bottles indicates a valid test. The absence ofany signs of growth in both the aerobic and anaerobic blood culturebottles, as well as no organisms seen on gram stain indicates a negativeblood culture. The presence and identification of microorganism(s) ineither the aerobic or anaerobic blood culture bottle indicates of apositive blood culture; this typicall is due to a bacteremic state.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

I claim:
 1. An embryonic porcine pancreatic cell, which, in unmodifiedform, has at least one antigen on the cell surface which is capable ofstimulating an immune response against the cell in a xenogeneic subject,wherein the antigen on the cell surface is altered to inhibit rejectionof the cell when introduced into the xenogeneic subject.
 2. The porcinepancreatic cell of claim 1, wherein the antigen on the cell surfacewhich is altered is an MHC class I antigen.
 3. The porcine pancreaticcell of claim 2, which is contacted prior to introduction into axenogeneic subject with at least one MHC class I antibody, or fragmentor derivative thereof, which binds to the MHC class I antigen on thecell surface but does not activate complement or induce lysis of thecell.
 4. The porcine pancreatic cell of claim 3, wherein the MHC class Iantibody is an anti-MHC class I F(ab')₂ fragment.
 5. The porcinepancreatic cell of claim 4, wherein the MHC class I F(ab')₂ fragment isa F(ab')₂ fragment of a monoclonal antibody PT85.
 6. The porcinepancreatic cell of claim 1, which is obtained from an embryonic pigbetween about day 31 and about day 35 of gestation.
 7. The porcinepancreatic cell of claim 1, which is obtained from an embryonic pigwhich is essentially free from organisms or substances which are capableof transmitting infection and disease to a xenogeneic recipient of thecells.
 8. The porcine pancreatic cell of claim 7, which is obtained froman embryonic pig which is essentially free from at least one organismselected from the group consisting of parasites, bacteria, mycoplasma,and viruses.
 9. An isolated population of embryonic porcine pancreaticcells which, in unmodified form, have at least one antigen on thesurface of the cells which is capable of stimulating an immune responseagainst the cells in a xenogeneic subject, wherein the antigen on thesurface of the cells is altered to inhibit rejection of the cells whenintroduced into the xenogeneic subject.
 10. The isolated population ofporcine pancreatic cells of claim 9, wherein the cells are in the formof islet-like aggregates.
 11. The isolated population of porcinepancreatic cells of claim 9, wherein the cells are obtained from anembryonic pig between about day 31 and about day 35 of gestation. 12.The isolated population of porcine pancreatic cells of claim 9, whereinthe cells are obtained from an embryonic pig which is essentially freefrom organisms or substances which are capable of transmitting infectionand disease to a xenogeneic recipient of the cells.
 13. The isolatedpopulation of porcine pancreatic cells of claim 12, wherein the cellsare obtained from an embryonic pig which is essentially free from atleast one organism selected from the group consisting of parasites,bacteria, mycoplasma, and viruses.
 14. A cell culture comprising apopulation of embryonic porcine pancreatic cells which, in unmodifiedform, have at least one antigen on the surface of the cells which iscapable of stimulating an immune response against the cells in axenogeneic subject, wherein the antigen on the surface of the cells isaltered to inhibit rejection of the cells when introduced into thexenogeneic subject.